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Glycosylation
plays an important role in many specific biological
functions, including immune defense, fertilization,
viral replication, parasitic infection, cell growth,
inflammation, and cell-cell adhesion. In addition,
glycosylation is the most versatile and one of the most
abundant of all co- and posttranslational
modifications.1 Procognia and QIAGEN have developed a
novel technology that provides a detailed
characterization of protein glycan moieties in a
fraction of the time required for conventional HPLC-based
methods of glycoanalysis.
Summary
Qproteome
GlycoArray technology (QGA) provides researchers with a
rapid and
simple kit-based
method for analysis of the glycosylation moieties of
glycoproteins produced in mammalian cells. QGA is based
on an array containing a set of 24 lectins with
overlapping specificities, which have been characterized
using a large dataset of carefully chosen,
well-characterized glycoproteins. Lectins with
differing, defined glycan-binding specificities are
spotted on the surface of an array. The array is probed
with a glycoprotein, washed to reduce background, and
visualized using a microarray scanner. Bound
glycoproteins can be visualized by direct fluorescent
labeling of the glycoprotein target(s), biotinylation of
the target(s) followed by detection with fluorescent
streptavidin, or by using a fluorescently labeled
protein-specific antibody (Fig. 1). After scanning the
array, fluorescent signals are evaluated using Qproteome
GlycoArray Analysis Software.
Binding
of a glycoprotein to the array results in a
characteristic “fingerprint” that is sensitive to
changes in the protein’s glycan composition (Fig. 2).
Though the fingerprint
is a sensitive tool for comparing differences in
glycosylation among samples of the same protein, it is
not a direct readout of the glycan structures in each
sample. To give a profile of the glycan structures,
fingerprint signals are automatically deconvoluted by a
set of proprietary algorithms.
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1. Glycoprotein is applied to the lectin array (A) and
its glycans are recognized by the arrayed lectins (B).
Binding is detected using an antibody probe (C), or
direct pre-labeling of the protein. After binding,
detection, and deconvolution, the relative fluorescence
of the array spots (D) provides a qualitative and
semi-quantitative analysis of the glycan epitopes
detected on the glycoprotein sample. |
The
method is simple and faster than conventional methods
that rely on chromatographic and mass spectrometric
techniques, as there is no need for purification or
time-consuming sample preparation steps.
Introduction
Glycosylation
results from sugar residues added to a protein backbone
that form a glycoprotein, which affects the stability of
protein conformation, clearance rate, protection from
proteolysis, and improves protein solubility.2–5
Glycosylation plays an important role in vivo in many
biological functions, including immune defense,
fertilization, viral replication, parasitic infection,
cell growth, inflammation, cell-cell adhesion, and
oncogenic transformation.6-13 In vitro, a protein’s
pattern of glycosylation depends on many factors;
including the type of cell producing the glycoprotein,
nutrient concentrations, pH, cell density, and age.
Different
cell lines and different fermentation conditions can
produce significantly different glycosylation patterns.
Glycosylation sites on glycoproteins commonly display
micro-heterogeneity;
they can be fully or partially occupied by
structurally diverse oligosaccharides. Mammalian
glycoprotein oligosaccharides are commonly built from a
limited number of monosaccharides, but their structural
diversity is vast, mainly because they often form
complex branching patterns. Glycosylation is not
template-driven, and is currently impossible to predict.
In
living cells, proteins are glycosylated by the actions
of a series of glycosidases and glycosyltransferases
that act sequentially on the growing glycan as it passes
through the lumen of the endoplasmic reticulum (ER) and
the Golgi apparatus. The various enzymatic reactions may
not all reach completion, therefore a variety of glycan
structures are commonly attached at each glycosylation
site. Consequently, under a given set of conditions, a
different population of glycosylation forms may be
generated for a single type of protein.
A
protein with a defined glycan pattern is termed a
glycoform. The abundance of glycoforms within a cell is
affected by the intrinsic structural properties of the
individual protein, as well as the selection of
glycosylation enzymes available (their type,
concentration, kinetic characteristics,
compartmentalization, etc.). The enzymes available have
been shown to change according to changes in cell state,
such as oncogenic transformation.13
Aberrant
glycosylation has been observed in various disease
states such as inflammatory diseases and cancer. Despite
numerous studies that unequivocally demonstrate that
oncogenic transformation is accompanied by changes in
glycosylation,
elaborate structural characterization of these changes
and understanding of glycosylation’s precise role in
the transformation process remain elusive. A key
obstacle limiting such studies is the lack of a
technology that can analyze the glycan composition of
glycoproteins directly from in vivo samples.
Most
cell surface and secreted proteins are glycosylated with
either N-linked carbohydrates covalently attached
through the side chain amide of asparagines, or O-linked
carbohydrates covalently attached through the side-chain
hydroxyl groups of serine or threonine. All N-linked
glycans contain the pentasaccharide Man a1-6 (Man
a1-3)
Man b1-4 GlcNAc
b1-4 GlcNAc as a common core
(Fig. 3) and can be classified into three main
groups: oligomannose (high-mannose), complex types (bi-,
tri-, and tetra- antennary; pentaantennary structures
are rare), and hybrid type.
Oligomannose
structures contain only a-mannosyl residues attached to
the trimannosyl core. Complex-type structures contain no
mannose residues other than the trimannosyl core, but
contain antennae with N-acetylglucosamine. The
structural variation results from the various
monosaccharides, such as sialic acid and fucose, which
can be found in the antennae and in the core. Hybrid
structures combine the characteristics of both
oligomannose and complex glycans.
The complex-type glycans exhibit the greatest
diversity, arising from both the number of antennae and
the monosaccharides attached to the antennae; for
example, diverse linkages of fucose to either galactose
or N-acetylglucosamine are a common epitope.
Lectin-Based
Glycoanalysis
Lectins
are a family of carbohydrate-recognizing proteins that
are classified into a number of specificity groups based
on the monosaccharides for which they exhibit the
highest affinity.14 The primary specificity groups of
plant lectins include mannose/glucose, galactose/N-acetylgalactosamine,
N-acetylglucosamine, fucose, and sialic acid. Primary
specificity for other monosaccharides is very rare.
The
classification of lectins based on their preferential
binding to monosaccharides is useful to distinguish
groups with differing gross specificities, yet in many
cases gives a misleading picture of the real
specificity. The concentrations required for abolishing
binding by mono- or disaccharides are usually high
(association constants of 103–104
M-1) and their
specificity is relaxed, especially when compared to the
typical inhibitory concentrations of oligosaccharides or
complex glycans (association constants of 105–107
M-1)
whose binding specificity is tighter. This heightened
specificity may be explained by the occurrence of
extended lectin-binding sites that preferentially
accommodate oligosaccharides.
The
plant lectins used in Qproteome GlycoArrays cover a
broad range of specificities. The arrays carry a set of
24 lectins with overlapping specificities, which have
been characterized using a large dataset of carefully
chosen, well-characterized glycoproteins. Binding of a
glycoprotein to the array results in a characteristic
fingerprint that is highly sensitive to changes in the
protein’s glycan composition. Qproteome GlycoArrays
provide researchers with a rapid, simple kit-based
method for determining the pattern and relative
abundance of specific mammalian glycosylation epitopes
in a glycosylated protein. The analysis can be performed
on crude samples in growth media, thus eliminating the
need for time-consuming purification and sample
preparation steps.
The
Properties of Lectins Used in Qproteome GlycoArrays
The
lectins printed on the Qproteome GlycoArrays have been
chosen by analyzing a set of over 80 lectins, using a
large dataset of carefully chosen, well-characterized
glycoproteins, and a large set of enzymatically
synthesized glycovariants of these proteins. A
description of the glycan epitopes analyzed by the kit
and their quantification classification can be found in
Table I.
Characterization
of EPO Variants
Recombinant
human erythropoietin (rhEPO) is widely used for the
treatment of EPO-deficient anemias. In addition to its
well-known role in erythropoiesis, EPO also plays an
important protective role in the nervous system.
However, large amounts of EPO lead to potentially
harmful increases in red cell mass and to the production
of hyperreactive platelets, which can lead to
thrombosis. While red blood cell production needs the
continuous presence of EPO, a brief exposition is
sufficient for neuroprotection. Asialoerythropoietin (asialoEPO)—a
desialylated form of EPO—is rapidly cleared from the
blood and does not increase erythrocyte mass, but is
fully protective in animal models for stroke, spinal
cord injury, and peripheral neuropathy.15
Method
An
aliquot of 50 µM purified commercial rhEPO was
desialylated by application of 0.16 U/ml neuraminidase
for 16 hours at 37° C in PBS. 500 µl aliquots of a 0.5
µM solution of both intact and desialylated samples
were processed using Qproteome GlycoArray slides
following a protocol provided in the handbook. Briefly,
lectin slides were blocked for 1 hour using a supplied
blocking buffer, washed, and a 450 µl sample was
pipetted onto the slide. The slides were incubated for 1
hour at room temperature. After a short wash step, the
slides were incubated with a rabbit anti-EPO antibody
for 40 minutes, washed, and incubated with a FITC-labeled
goat anti-rabbit secondary antibody for 30 minutes.
After incubation, the slides were washed again, dried,
and scanned using a microarray scanner with a FITC (488
nm) laser with a 530-nm emission filter. The scanned
image was analyzed using software provided in the kit.
Results
Glycoanalysis
using Qproteome GlycoArrays clearly demonstrates the
difference between intact rhEPO and asialo-rhEPO. The
signals displayed in the glycan fingerprint can give
strong evidence of the difference between glycoprotein
samples. The fingerprint of intact rhEPO shows a very
strong signal for sialic acid (Fig. 2, blue bars). As
expected, this signal is absent in the asialo-rhEPO
fingerprint (Fig. 2, pink bars). The removal of sialic
acid in the asialoEPO sample is further indicated by the
increased strength of the terminal beta-galactose (Beta
Gal) signals, particularly in the terminal beta-galactose
lectin 2 (Beta Gal 2), which is absent in the intact
protein.
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2. “Glycan fingerprint” analysis of an intact rhEPO
and n desialylated rhEPO (asialo-rhEPO). Average
fluorescent signals from several replicate spots for
each of the 24 lectins were normalized to background
fluorescence. |
However,
the relative promiscuity (e.g., compared to
antibody-antigen interactions) of lectins for different
glycans and their isomers requires that the raw data be
deconvoluted using a complex algorithm before concrete
conclusions can be drawn about the abundance or presence
of the eight glycan classes listed in the final result
table (Table II). This
algorithm is based on deconvolution of signals from
several lectins with overlapping and/or complementary
specificities for each epitope, and requires that no
contradicting signals are present.
In this case, the difference between intact rhEPO
and asialoEPO is clearly demonstrated. Both samples
contain high levels of complex tri- and tetra- antennary
structures and low levels of bi-antennary structures.
The native sample is highly sialylated, whereas the
desialylated sample does not contain detectable levels
of sialic acid (Table II).
Discussion
The
different glycoforms of EPO have different properties
and effects in vivo. The serum half-life of native EPO
is greaterthan 200-fold higher than the desialylated
variant. Intact EPO induces erythropoiesis and has a
neuroprotective effect, whereas asialoEPO merely has a
neuroprotective effect. Presumably, these differences
are due to the difference in glycan groups on the
proteins’ surfaces.
With
the Qproteome GlycoArray Kit, analysis of the two rhEPO
glycoforms clearly indicates
differences in protein glycan makeup. In addition, the
Kit facilitates the investigation of relationships
between protein glycosylation and function. The
procedure provides semi-quantitative and qualitative
analysis for the major classes of glycan moieties, and
is therefore suitable for many applications in life
science research.
The
European Pharmacopeia requests isoelectric focusing (IEF)
and SDS-PAGE analysis for quality assurance of rhEPO and
other glycosylated protein therapeutics.16 Another
commonly used method for glycoanalysis is sequential
deglycosylation using specific enzymes followed by
HPLC.17 In contrast to such methods, GlycoArray analysis
provides a fast and convenient procedure.
We have
demonstrated that glycoanalysis using Qproteome
GlycoArray slides enables a detailed comparison of rhEPO
and its desialylated variant and provides comparable
information in a fraction of the time required by
currently used methods.
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3. Basic structure of N-linked glycans. Asn; asparagine,
Fuc; fucose, GlcNac; N-acetylglucosamine, Man; mannose,
Gal; galactose, NeuNAc; N-acetylneuraminic acid. |
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